Project Summary Much of our understanding of brain microcirculation comes from studies on arteriolar perfusion. Blood efflux through venules plays an equally important role in determining blood flow through the brain, since all blood entering the brain must exit via venules. The structure and function of cerebral venules can change dramatically during cerebrovascular disease. Preclinical and clinical studies have demonstrated marked alterations in venule tortuosity and vascular wall composition during Alzheimer?s disease and Alzheimer?s disease-related dementias. Compared to arterioles, the slower flow and distinct endothelial biology of venules makes them more susceptible to hemostasis, thrombosis, and immune cell adhesion during disease. Collectively, these factors point to venules as a site of vulnerability in cerebral perfusion that remains highly understudied. This project focuses on principal cortical venules (PCVs), a subset of venules that descend from the brain surface into the deepest layers of cortex and underlying white matter. Although PCVs are less common compared to smaller cortical venules, they extend massive, horizontally projecting branches in deeper tissues, suggesting a critical role in perfusion of deep cortex and adjacent white matter tracts. However, there exists almost no information on the structure, physiology and perfusion territories of PCVs. Cerebral white matter is particularly sensitive to blood flow deficit and degenerates in early stages of Alzheimer?s disease and Alzheimer?s disease-related dementias. Understanding the regulation of perfusion in and near white matter tracts will be critical in understanding the basis of this white matter degeneration. Our central hypothesis is that PCVs are the main drainage system for deep cortical layers and the underlying white matter. In Aim 1, we will test this hypothesis by using emergent deep in vivo two-photon imaging and three-photon imaging to measure how capillary flow is drained in cortical layer 6 and its adjacent white matter tract in the mouse brain, respectively. These activities will be performed in adult (3-9 months) and aged mice (18-24 months) to test a secondary hypothesis that age is associated with deterioration in PCV structure and function. In Aim 2, we will we will quantify the radius of cortical tissue dependent upon PCV drainage by measuring how photothrombotic occlusion of a single PCV affects flow into the cortex through neighboring penetrating arterioles. We will further use histology to assess the volume of hypoxic tissue in gray and white matter created by occlusion of single PCVs. This project is significant because it addresses the understudied topic of venular perfusion in white matter using novel in vivo imaging approaches. It further establishes an experimental foundation needed for future research on venular dysfunction as a mechanism of impaired cerebral blood flow and white matter degeneration in Alzheimer?s disease and Alzheimer?s disease-related dementias.